Tyrosine sulfation is an ubiquitous post-translational modification that occurs in essentially all animal cells containing a Golgi apparatus (Huttner, 1998, Ann. Rev. Physiol. 50: 363-376). As much as 1% of the tyrosine residues in an organisms total protein are capable of being sulfated. Tyrosine sulfation of proteins occurs in the trans-Golgi, and the modification appears to be involved in intracellular transport, proteolytic processing and alteration of biological activity. The ability to detect, isolate and differentiate sulfotyrosine-containing proteins would therefore prove extremely valuable. However, no antibody capable of discriminating tyrosine-sulfated proteins from non-sulfated proteins has been described. More particularly, tyrosine sulfation has been found to drive extracellular protein-protein interactions. The archetypal example is the interaction between PSGL-1 and P-selectin, in which PSGL-1's sulfated tyrosine residues are positioned to form hydrogen bonds with P-selectin. Additionally, the tyrosine-sulfated chemokine receptors CCR5, CXCR4, and CCR2 rely on their sulfate groups to increase binding affinity for their chemokines, and in the case of CCR5 are exploited by HIV to mediate infection.
Antibodies against posttranslational modifications have proven to be vital tools for functional studies. For example, an antibody against nitrotyrosine was recently used to identify proteins which are nitrated during the inflammatory response, and antibodies against tyrosine phosphate have been widely used for decades. The obvious utility of an antibody against tyrosine sulfate has led a number of laboratories to pursue this important tool (Bundgaard et al., 2002, Analysis of Tyrosine-O-Sulfation, Methods in Molecular Biology Posttranslational Modification of Proteins, pp. 223-239). While an antibody whose binding epitope includes tyrosine sulfate has been reported (Snapp et al., 1998, Blood 91: 154-164), there are no reports of an antibody that recognizes only a sulfated tyrosine residue, despite numerous unsuccessful immunization attempts to derive antibodies recognizing sulfated tyrosines. One explanation for these failures is that the presence of sulfated tyrosine residues in many secreted and membrane-bound proteins has led vertebrate immune systems to become tolerant of the modification, rendering standard immunization-based antibody generation methodologies useless.
One way to overcome the limitations of intact immune systems in the generation of specific antibodies against non-immunogenic targets is to use phage antibody libraries rather than immunization. In this technique large numbers (≧109) of different antibodies are displayed on the surface of filamentous phage and specific binders are selected on the basis of their binding abilities to target antigens. The fact that this technology is completely in vitro, using either natural rearranged or synthetic V genes, overcomes the intrinsic biases of the immune system. Although phage antibodies have been selected against large numbers of different polypeptide and chemical targets, including specific peptides, there have been no descriptions of the use of this technology to select antibodies against post-translational modifications.
There is therefore a need for antibodies capable of specifically recognizing tyrosine-sulfated proteins and capable of distinguishing between sulfated and non-sulfated proteins. The present invention addresses this need.